MYO5C Antibody

What is MYO5C and what cellular processes is it involved in?

MYO5C (myosin VC) is a motor protein predominantly expressed in epithelial and glandular tissues where it plays a critical role in secretory processes. The canonical human protein consists of 1742 amino acid residues with a molecular mass of approximately 202.8 kDa . Structurally, MYO5C comprises two heavy chains, each containing a motor domain, an elongated neck domain with six IQ motifs that bind calmodulin or myosin light chains, a coiled-coil dimer-forming region, and a carboxyl-terminal globular tail domain . Functionally, MYO5C is involved in transferrin trafficking and likely powers actin-based membrane trafficking in physiologically important tissues . While MYO5C is classified as a low duty cycle motor, when two or more MYO5C-heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments .

In which tissues and cell types is MYO5C predominantly expressed?

MYO5C is expressed primarily in non-neuronal tissues, with particularly high abundance in epithelial and glandular tissues . Specific expression has been documented in pancreas, prostate, mammary glands, stomach, colon, and lung . Immunohistochemical studies have revealed particularly high levels of MYO5C in insulin-producing β-cells located within the pancreatic islets of Langerhans . This tissue-specific expression pattern is significant for researchers designing experiments involving MYO5C, as it informs the selection of appropriate cell lines and tissue samples.

What are the critical considerations when selecting a MYO5C antibody?

When selecting a MYO5C antibody, researchers should consider:

The immunogen information is particularly important, as antibodies raised against different regions of MYO5C may perform differently in various applications. Additionally, it is advisable to select antibodies that have been validated specifically for your intended application and tissue/cell type of interest .

What are the optimal conditions for using MYO5C antibodies in Western blot analysis?

For optimal Western blot results with MYO5C antibodies, consider the following protocol adjustments:

• Sample Preparation: Given the high molecular weight of MYO5C (~202.8 kDa), use lower percentage gels (6-8%) for better resolution.

• Transfer Conditions: Employ longer transfer times or semi-dry transfer systems optimized for high molecular weight proteins.

• Antibody Dilution: A typical working dilution range is 1:500-1:2000, but this should be optimized for each specific antibody .

• Molecular Weight Considerations: Be prepared to observe bands at approximately 300 kDa rather than the calculated 202 kDa due to post-translational modifications or structural factors . This discrepancy is common with MYO5C and should not be interpreted as non-specific binding.

• Control Selection: Include positive controls from tissues known to express MYO5C highly (pancreas, prostate, mammary tissue) and negative controls from tissues with minimal expression .

The observed molecular weight of MYO5C can differ from the expected size due to various factors affecting mobility rates in gel electrophoresis, including different modified forms of the protein appearing simultaneously .

How should experimental designs address the study of MYO5C's role in membrane trafficking?

To effectively study MYO5C's role in membrane trafficking, particularly in transferrin trafficking, implement the following methodological approaches:

• Co-localization Studies: Design immunofluorescence experiments to visualize MYO5C alongside transferrin receptors or other trafficking markers using confocal microscopy.

• Functional Assays: Develop transferrin uptake and recycling assays in cells with normal, depleted, or overexpressed MYO5C to quantify trafficking efficiency.

• Molecular Inhibition Approaches: Utilize pentabromopseudilin (PBP), which inhibits MYO5C motor function with a half-maximal concentration of 280 nM by binding near the actin and nucleotide binding site .

• Tropomyosin Interactions: Investigate how cofilaments of actin and tropomyosin isoforms (Tpm1.6, Tpm1.8, or Tpm3.1) affect the actin-activated ATPase and motile activity of MYO5C .

• Live-Cell Imaging: Implement time-lapse microscopy of fluorescently-tagged MYO5C to track its movement along actin filaments in relation to membrane vesicles.

This multi-faceted approach allows for comprehensive analysis of both the molecular mechanisms and cellular consequences of MYO5C function in membrane trafficking.

What purification methods are recommended for MYO5C protein studies?

When purifying MYO5C for functional studies, consider these methodological approaches:

• Recombinant Production Systems: Utilize co-expression systems with calmodulin (CaM) alone or with CaM plus essential and regulatory light chains (Myl6 and Myl12b) to produce functional MYO5C-HMM constructs .

• Affinity Purification: Implement affinity chromatography techniques, as demonstrated in successful purification of MYO5C antibodies .

• Quality Control Assessment: Verify purified protein functionality through actin-activated ATPase assays and motility assessments before proceeding with further studies .

• Storage Conditions: Store purified proteins at -20°C in buffers containing glycerol (typically 50%) to maintain stability, avoiding repeated freeze-thaw cycles .

These approaches enable the isolation of functionally intact MYO5C for detailed biochemical and biophysical characterization.

Why might I observe discrepancies between calculated and observed molecular weights of MYO5C in Western blots?

The discrepancy between calculated (202 kDa) and observed (approximately 300 kDa) molecular weights of MYO5C is a common phenomenon with several potential explanations:

• Post-translational Modifications: Phosphorylation, glycosylation, or other modifications can significantly alter protein migration patterns.

• Protein Conformation: Native structural features of MYO5C may affect its electrophoretic mobility.

• Multiple Isoforms: The presence of different isoforms (up to two have been reported for MYO5C) may result in multiple bands or bands of unexpected sizes .

• Sample Preparation Conditions: Incomplete denaturation or reduction can affect migration patterns.

As noted in the technical literature: "Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

To address this issue, researchers should verify antibody specificity through additional controls and consider using multiple antibodies targeting different epitopes of MYO5C to confirm results.

How can I validate the specificity of MYO5C antibody staining in immunohistochemistry?

To validate MYO5C antibody specificity in immunohistochemistry (IHC), implement the following methodological steps:

• Positive Tissue Controls: Include tissues known to express high levels of MYO5C (pancreas, prostate, mammary, stomach, colon, and lung) as positive controls .

• Negative Tissue Controls: Include neuronal tissues which typically have low MYO5C expression .

• Peptide Competition Assays: Pre-incubate the antibody with the immunogen peptide to demonstrate that staining is blocked by specific competition.

• Correlation With Other Detection Methods: Validate IHC findings with in situ hybridization or RT-PCR to confirm mRNA expression patterns.

• Cross-Validation With Multiple Antibodies: Use antibodies targeting different epitopes of MYO5C to confirm staining patterns.

• Knockout/Knockdown Controls: If available, include samples from MYO5C knockout models or cells with MYO5C knockdown as negative controls.

The importance of proper validation is highlighted by the finding of particularly high levels of MYO5C in insulin-producing β-cells within pancreatic islets of Langerhans , demonstrating the cell-type specificity that should be observed with a specific antibody.

What are common technical challenges when using MYO5C antibodies in functional studies?

Researchers commonly encounter these technical challenges when using MYO5C antibodies in functional studies:

• Background Signal: High background can occur due to non-specific binding, particularly in tissues with abundant extracellular matrix.

  • Solution: Optimize blocking procedures and include appropriate controls to distinguish specific from non-specific signals.

• Epitope Masking: Protein interactions or conformational changes may mask antibody epitopes during functional studies.

  • Solution: Consider using antibodies targeting different regions of MYO5C, such as those directed at AA range 850-930 versus recombinant fusion protein-derived antibodies .

• Antibody Interference: Antibodies may interfere with MYO5C function when used in live-cell studies.

  • Solution: Consider alternative approaches such as GFP-tagged MYO5C constructs for functional studies.

• Cross-Reactivity: Possible cross-reactivity with other myosin V family members (MYO5A, MYO5B).

  • Solution: Perform parallel experiments in tissues with differential expression of myosin V isoforms to confirm specificity.

• Quantification Challenges: Variability in staining intensity across samples.

  • Solution: Implement standardized protocols, include calibration samples, and use digital image analysis for quantification.

These challenges underscore the importance of rigorous experimental design and appropriate controls when conducting functional studies with MYO5C antibodies.

How can I investigate the regulatory mechanisms affecting MYO5C motor function?

To investigate regulatory mechanisms of MYO5C motor function, implement the following experimental approaches:

• Tropomyosin Interaction Studies: Examine how cofilaments of actin and different tropomyosin isoforms (Tpm1.6, Tpm1.8, or Tpm3.1) alter the maximum actin-activated ATPase and motile activity of recombinant MYO5C constructs .

• Small Molecule Modulator Studies: Utilize pentabromopseudilin (PBP), which binds near the actin and nucleotide binding site (calculated ΔG of −18.44 kcal/mol) and inhibits MYO5C motor function with a half-maximal concentration of 280 nM .

• Light Chain Composition Analysis: Compare the functional properties of MYO5C-HMM co-produced with calmodulin alone versus MYO5C-HMM with calmodulin plus essential and regulatory light chains (Myl6 and Myl12b) .

• Single-Molecule Motility Assays: Employ total internal reflection fluorescence (TIRF) microscopy to visualize the processive movement of linked MYO5C-HMM molecules along actin filaments under various regulatory conditions.

• Structural Biology Approaches: Implement cryo-electron microscopy or X-ray crystallography to determine how regulatory factors induce conformational changes in MYO5C.

These approaches provide complementary insights into the complex regulatory mechanisms controlling MYO5C function in different cellular contexts.

What methodologies enable the study of MYO5C's role in specialized secretory pathways?

To investigate MYO5C's role in specialized secretory pathways, particularly in epithelial and glandular tissues, implement these methodological approaches:

• Tissue-Specific Expression Analysis: Conduct immunohistochemical staining to determine MYO5C distribution and localization in endothelial and endocrine cells from appropriate tissue samples .

• Secretory Cargo Tracking: Develop assays that track the movement of specific secretory cargo (e.g., insulin granules in pancreatic β-cells) in relation to MYO5C localization and activity.

• Domain-Function Analysis: Create and express truncated or mutated versions of MYO5C to determine which domains (motor, IQ motifs, coiled-coil, or tail) are critical for specific secretory functions.

• Interaction Proteomics: Implement immunoprecipitation followed by mass spectrometry to identify MYO5C-interacting proteins within secretory pathways.

• Live-Cell Dynamics: Utilize high-resolution live imaging of fluorescently tagged MYO5C in polarized epithelial cells to capture its dynamics during secretory vesicle transport.

These approaches have revealed significant MYO5C expression in insulin-producing β-cells within pancreatic islets of Langerhans , suggesting important roles in regulated insulin secretion that warrant further investigation.

How does MYO5C compare functionally with other myosin V family members?

The functional comparison between MYO5C and other myosin V family members reveals important distinctions:

To study these functional differences:

• Comparative Motility Assays: Conduct side-by-side in vitro motility assays with purified MYO5A, MYO5B, and MYO5C to directly compare motor properties.

• Isoform-Specific Knockdown/Rescue: Perform knockdown of endogenous myosin V isoforms followed by rescue with specific isoforms to determine functional redundancy or specialization.

• Chimeric Protein Analysis: Create chimeric proteins swapping domains between MYO5C and other myosin V isoforms to identify domains responsible for specific functional properties.

• Differential Inhibitor Sensitivity: Examine the relative sensitivity of different myosin V isoforms to inhibitors like pentabromopseudilin to identify isoform-specific regulatory mechanisms .

These comparative approaches reveal that while MYO5C shares structural similarities with other myosin V family members, its tissue distribution and functional properties suggest specialized roles in secretory and membrane trafficking pathways.

What is the potential significance of MYO5C in endocrine cell function and disease?

The high expression of MYO5C in insulin-producing β-cells within pancreatic islets of Langerhans suggests potentially significant roles in:

• Regulated Insulin Secretion: MYO5C may facilitate the trafficking of insulin granules along actin filaments to the plasma membrane during glucose-stimulated insulin secretion.

• β-Cell Dysfunction in Diabetes: Alterations in MYO5C expression or function could potentially contribute to impaired insulin secretion in diabetes mellitus.

• Secretory Granule Biogenesis: MYO5C may participate in the formation and maturation of insulin-containing secretory granules within the trans-Golgi network.

To investigate these possibilities, researchers should consider:

• Developing β-cell specific MYO5C knockout or knockdown models to assess effects on insulin secretion

• Examining MYO5C expression and localization in β-cells from diabetic versus non-diabetic donors

• Utilizing advanced imaging techniques to track MYO5C-mediated granule movement in response to secretagogues

This emerging research direction could significantly advance our understanding of the molecular machinery underlying regulated secretion in endocrine cells and potentially identify new therapeutic targets for secretory disorders.

How do post-translational modifications regulate MYO5C function?

The significant discrepancy between calculated (202 kDa) and observed (300 kDa) molecular weights of MYO5C suggests extensive post-translational modifications (PTMs) that may regulate its function. To investigate this:

• PTM Mapping: Employ mass spectrometry to identify and map phosphorylation, glycosylation, ubiquitination, and other modifications on MYO5C.

• Site-Directed Mutagenesis: Generate MYO5C constructs with mutations at putative modification sites to assess functional consequences.

• Regulatory Enzyme Studies: Identify kinases, phosphatases, and other enzymes that modify MYO5C and examine how their inhibition or activation affects MYO5C localization and function.

• Domain-Specific Modification Analysis: Determine whether modifications cluster in specific functional domains (motor, neck, tail) and correlate with distinct functional outcomes.

• Stimulus-Dependent Modification: Investigate how cellular stimuli relevant to secretory processes alter the PTM profile of MYO5C.

Understanding the "PTM code" of MYO5C could provide critical insights into the molecular mechanisms regulating its activity in different cellular contexts and potentially reveal new approaches to modulate its function in disease states.

What novel technologies are advancing our understanding of MYO5C dynamics?

Emerging technologies are providing unprecedented insights into MYO5C dynamics:

• Single-Molecule Biophysics: High-resolution optical trapping techniques allow precise measurement of forces generated by individual MYO5C molecules, revealing how factors like tropomyosin modulate its mechanical properties .

• Cryo-Electron Microscopy: Advanced structural determination of MYO5C complexes in different nucleotide states provides insights into conformational changes during the mechanochemical cycle.

• Molecular Docking Studies: Computational approaches predict how small molecules like pentabromopseudilin bind to MYO5C with high affinity (calculated ΔG of −18.44 kcal/mol), enabling rational design of isoform-specific modulators .

• Genome Editing in Primary Cells: CRISPR-Cas9 editing of endogenous MYO5C in primary epithelial and glandular cells allows study of its function in physiologically relevant contexts.

• Super-Resolution Microscopy: Techniques like STORM and PALM enable visualization of MYO5C dynamics at nanometer resolution in living cells, revealing its organization at membrane trafficking hotspots.

These technological advances provide complementary approaches to understand MYO5C function across scales from single molecules to intact tissues, accelerating discovery of its roles in normal physiology and disease states.